Abstract Hydrogen sulfide (H2S) is toxic at high concentrations but beneficial at low, eliciting profound effects on physiological functions ranging from cardioprotection to hippocampal long-term potentiation, to protection against intestinal inflammation. Its toxicity is averted by the robust activity of a mitochondrial sulfide oxidation pathway, which feeds into the electron transfer chain, linking H2S to energy metabolism. H2S is the product of three enzymes in the sulfur network of which two, cystathionine ?-synthase (CBS) and ?-cystathionase (CSE), reside in the cytoplasmic transsulfuration pathway while the third, mercaptopyruvate sulfurtransferase (MST), is involved in cysteine catabolism and is primarily mitochondrial in location. Studies in our laboratory have provided detailed insights into the kinetic and chemical mechanisms of the human enzymes revealing a suprising laxity in substrate and reaction specificity particularly for CBS and CSE. Together, the transsulfuration enzymes catalyze at least eight H2S generating reactions in addition to the two canonical cysteine producing reactions, raising the obvious question of how substrate and reaction choices are regulated in a cellular milieu. In the next cycle, we propose to elucidate fundamental mechanisms of regulation of H2S synthesis in normal and disease states by addressing the following specific aims: (i) Elucidate allosteric regulation in CBS, which we have recently discovered uses a unique mechanism via its heme cofactor, to regulate the choice of substrate for CSE, the next enzyme in the pathway. For this, we will exploit an enigmatic set of pathogenic CBS mutations that retain normal activity but exhibit faulty allosteric regulation that is exerted over 50 of protein terrain. (ii) Elucidate regulation of H2S synthesis by CSE in the context of angiogenesis in which NO and CO play a role, and which we can now connect to CSE via the NO and CO-sensistive heme- regulated switch in CBS. We will also develop CSE inhibitors by optimizing lead compounds that show specificity towards CSE, the major H2S producer in the peripheral system. (iii) We will elucidate the role of MST in proliferation and cellular bioenergetics, building on our unpublished studies, which reveal significant up- regulation of MST in colon cancer. We will also assess the impact of MST on H2S signaling using a newly developed, sensitive, and specific persulfide tagging method for proteomic analysis. The impact of our proposed studies will be both fundamental (i.e. elucidating mechanims of allosteric regulation at the level of individual enzymes and in the pathway), and medical (i.e. understanding the biochemical basis of failure of disease-causing CBS mutations, developing CSE inhibitors with therapeutic potential and identifying changes in the persulfide proteome in colon cancer associated with metabolic reprogramming).